Rapamycin enhances VEGFA and VSMC contractile protein expression in MSCs via mTORC1 inhibition

Abstract

IntroductionIntracranial aneurysm (ICA) rupture is the most common cause of non-traumatic subarachnoid hemorrhage, a devastating type of stroke. Vascular smooth muscle (VSMC) and endothelial cell (EC) dysfunction and death play important roles in the etiology of ICA formation, rupture and treatment failure. Mesenchymal stem cells (MSCs) have been extensively investigated for their therapeutic potential in vascular diseases. The mammalian target of rapamycin (mTOR) is a key regulatory pathway involved in cellular functions controlling intracellular anabolic and regulatory processes. Rapamycin, a specific mTOR complex 1 inhibitor, is widely used in the clinical management of cardiac and vascular pathologies. In this study, we explored MSCs potential to express VSMC and EC markers under the influence of rapamycin. MethodsHuman MSCs were treated with rapamycin for 2, 5, and 10 days, and cell death and proliferation determined by MTT analysis. Protein expression and phosphorylation levels were determined by western blot analysis. Calcein AM and PI staining was used to determine cell viability, and morphology. ResultsMTT and Calcein AM analysis demonstrated that prolonged rapamycin treatment did not affect MSC viability but was found to reduce MSC proliferation and cause a nearly threefold increase in cell size compared to controls. Rapamycin treatment increased expression of the VSMC protein markers, -SMA, and transgelin and was required to maintain elevated expression levels both proteins. In contrast, selective inhibition of mTORC2 with JR-AB2-01 caused a decrease in -SMA and trangelin expression, suggesting mTORC1 regulation of -SMA and transgelin expression, rather than mTORC2. Rapamycin treated MSC were also found to induce cell sprouting between adjacent cells. MSCs were found not to express endothelial protein markers under either basal conditions or following rapamycin treatment. However, rapamycin treatment was found to increase VEGFA expression, a known promoter of angiogenesis. ConclusionRapamycin has the potential to increase VSMC protein expression in MSCs by its action on mTORC1. The ability of rapamycin to increase VEGFA expression in MSCs could allow for a novel MSC-based therapeutic intervention in cerebral aneurysm management which could aid in ICA healing and reducing rupture risk.